Gene Cloning And Construction Of Over-expression Vectors Of MYB Genes In Rose

Rose (Rosa rugosa Thunb.) is an important ornamental plant of rosaceous, with beautiful and fragrant flowers. Attributed to its colorful and scented flowers, cold and drought resistance, it has huge potential for landscaping to address environmental degradation today. In addition, rose essential oil extracting from its petals, known as "liquid gold", is very famous and valuable in the world. Nevertheless, until now people's study on the rose main focus on the cultivation and management. It is possible to improve rose's horticultural traits by means of modern developing biotechnology.Recently, it has became a focus of research to use MYB transcription factors for improving horticultural traits of ornamental plants, such as flower color, scent, etc.In our study, we collected rose mixed petals and sended them to BGI for RNA-Seq. Meanwhile, total RNA of high-quality from rose mixed petals were extracted, and we reversed it to cDNA.We screened thirteen ESTs of MYB genes, designed primers according to ESTs, and cloned the gDNA fragments of MYB genes from rose genomic DNA. We used TAIL-PCR stategy to isolate the target DNA segments flanking known sequences, until got the two UTRs. At the last, we designed primers according to UTR of MYB genes, cloned the gDNA and cDNA sequences.Of those isolated MYB genes, six were used to construct of over-expression vectors, aimed to illustrate their putative fuction. The main research results of this study as follows:1. Firstly, we screened thirteen ESTs of MYB genes from the results of RNA-Seq, and cloned the entire genes by means of combination of general PCR and TAIL-PCR. Of the thirteen genes, two genes, RrMYB8 and RrMYB10, were belonged to R1/R2 MYB genes. There were ten R2R3-MYB genes cloned in this study, including RrMYB1,RrMYB2,RrMYB3,RrMYB5,RrMYB6,RrMYB7,RrMYB9,RrMYB11,RrMYB12 and RrMYB13. One, RrMYB4, was belonged to R1R2R3-MYB genes. The protein sequences alignment of the reported MYB genes and that in this study indicates that MYB genes cloned in this study have structural features of MYB genes, such as DNA-binding domain. So we preliminary confirmed the gene cloned in this study belonged to MYB family.2. Secondly, we constructed six over-expression vectors of MYB genes. In which, the cDNA of RrMYB8, a R1/R2-type MYB gene, was to construct the pCAMBIA2300s-RrMYB8 binary vector. We selected five R2R3-MYB genes, cDNA of RrMYB1, gDNA of RrMYB2, gDNA of RrMYB5, cDNA of RrMYB6 and cDNA of RrMYB7, for construction of PMV-RrMYB1, pCAMBIA2300s-RrMYB2, PMV-RrMYB5, PMV-RrMYB6 and PMV-RrMYB7. In addition, recombinant plasmids were transformed into agrobacterium EHA105.